Sorbent selectivity, HPLC

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

The most common analytical methods used were gas chromatography, HPLC, AA spectrophotometry, polarography, colorimetry, and potentiometry with ion-selective electrodes. In this study GC/MS and other more expensive instrumentation were avoided. If sorbent tubes could not be used for gaseous substances, then the less desirable miniature bubblers or impingers were considered. Although these devices are inconvenient they were often used because no better alternatives were available. Bags were used in a few cases where the analyte could not be retained on a sorbent because of volatility and a small tendency to sorb. Filters were used for particulates. Combinations of collection devices were used if we felt that both particulates and vapor might be present in the analyte. 

Traditional (i.e., non-MIP) SPE sorbents are similar to HPLC stationary phases. The advantages of many of these materials are that they are widely available, well characterized, have high binding capacity, and show linear adsorption behavior. One may observe that just a few types are used in the majority of sample preparations, i.e., these materials are quite generic, and it is the wash and elution step which is varied according to the application. The generic nature of these materials is also a drawback because it reflects their limited selectivity. 

Selected data published by Patsias and Papadopoulou-Mourkidou [114] illustrate sorption s dependence on sample volume (Figure 2.36). Their research pursues development of an automated online SPE-HPLC methodology for analysis of substituted anilines and phenols. Recovery (%) was measured for numerous compounds on various polymeric sorbents, but the only data presented here are those in which a styrene-divinylbenzene polymeric sorbent was used for analysis of aniline, phenol, 4-nitroaniline, and 4-nitrophenol. Aqueous sample volumes of 5, 10, 25, 50, 75, 100, 125, and 150 mL were acidified to pH 3 before SPE. 

On the other hand, the lack of internal pore structure with micropellicular sorbents is of distinct advantage in the analytical HPLC of biological macromolecules because undesirable steric effects can significantly reduce the efficiency of columns packed with porous sorbents and also result in poor recovery. Furthermore, the micropellicular stationary phases which have a solid, fluid-impervious core, are generally more stable at elevated temperature than conventional porous supports. At elevated column temperature the viscosity of the mobile phase decreases with concomitant increase in solute diffusivity and improvement of sorption kinetics. From these considerations, it follows that columns packed with micropellicular stationary phases offer the possibility of significant improvements in the speed and column efficiency in the analysis of proteins, peptides and other biopolymers over those obtained with conventional porous stationary phases. In this paper, we describe selected examples for the use of micropellicular reversed phase... 

In the case of gel permeation or size-exclusion HPLC (HP-SEC), selectivity arises from differential migration of the biomolecules as they permeate by diffusion from the bulk mobile phase to within the pore chambers of the stationary phase. Ideally, the stationary phase in HP-SEC has been so prepared that the surface itself has no chemical interaction with the biosolutes, with the extent of retardation simply mediated by the physical nature of the pores, their connectivity, and their tortuosity. In this regard, HP-SEC contrasts with the other modes of HPLC, where the surfaces of the stationary phase have been deliberately modified by chemical procedures by (usually) low molecular weight compounds to enable selective retardation of the biosolutes by adsorptive processes. Ideally, the surface of an interactive HPLC sorbent enables separation to occur by only one retention process, i.e., the stationary phase functions as a monomodal sorbent. In practice with porous materials, this is rarely achieved with the consequence that most adsorption HPLC sorbents exhibit multimodal characteristics. The retention behavior and selectivity of the chromatographic system will thus depend on the nature and magnitude of the complex interplay of intermolecular forces... 

Such approaches underpin the current popularity of RP-HPLC procedures for the purification of synthetic or recombinant polypeptides at the production scale, or analogous approaches employed in the HP-IEX of commercially valuable proteins. However, in some cases when linear scale-up methods are applied to higher molecular weight polypeptides or proteins, their biological activity may be lost due to unfavorable column residency effects and sorbent surface area dependencies. It is thus mandatory that the design and selection of preparative separation system specifically address the issues of recovery of bioactivity. Often some key parameters can be easily controlled, i.e., by operating the preparative separation at lower temperatures such a 4°C, or by minimizing column residency times. 

As noted, and as detailed in Table 2, a large variety of stationary-phase and mobile-phase factors influence the selectivity, recovery, and stability of proteins and other biomacromolecules in the adsorptive modes of HPLC. Batch adsorption pilot experiments provide an expedient approach to ascertain the effect of many parameters, such as the pH, nature, and concentration of organic solvent or ionic additives in the mobile phase, the temperature- or the static-binding capacity with a defined sorbent. Similarly, the influence of... 

All of these effects impact on the loading capacity of a particular HPLC sorbent, which can thus exhibit subtly different selectivity-capacity dependencies with different classes of polypeptides and proteins. Such behavior has been documented8,78,160,150,227-230 for enzymes and other proteins in a variety of studies. For example, when conformational reordering of a protein structure occurs in both the mobile phase and the stationary phase, this will... 

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